Compositions for lessening oxidative stress

ABSTRACT

The present invention provides an antioxidative composition with high safety, which is capable of lessening oxidative stress due to active oxygen species, free radicals, or the like in vivo, thereby preventing the occurrence or worsening of a disease.  
     It was confirmed that by using a composition containing oxidized coenzyme Q and/or reduced coenzyme Q, the amount of urinary 8-hydroxydeoxyquanosine can be decreased in normal or diabetic animals. Also, in histopathological research of the spleens of diabetic rats, it was confirmed that spleen tissue denaturation caused by oxidative stress can be prevented. It was thus found that oxidative stress in vivo can be lessened.  
     According to the present invention, oxidative stress in vivo can be lessened by using a composition containing coenzyme Q as an active ingredient.

TECHNICAL FIELD

[0001] The present invention relates to an antioxidative composition capable of lessening oxidative stress in vivo.

BACKGROUND ART

[0002] In recent years, adverse effects of oxidative stress in vivo, i.e., active oxygen species (hydroxy radicals, alkoxy radicals, hydroperoxy radicals, peroxy radicals, iron-oxygen complexes, superoxides, hydrogen peroxide, hydroperoxides, singlet oxygen, and ozone) or free radicals (lipid radicals and the like) on diseases have been made clear. The most popular theory is that arterial sclerosis is caused by oxidation of low-density lipoproteins (LDL) in the plasma. This theory is that oxidized LDL, i.e., LDL undergoing lipid oxidation due to oxidative stress, causes foaming of macrophages to cause arterial sclerosis. Many researchers have given affirmative reports. Also, the theory is strongly supported by the fact that probucol as a cholesterol decreasing medicine exhibiting an antioxidative activity exhibits effectiveness for arterial sclerosis. Besides arterial sclerosis, the effect of oxidative stress on carcinogenesis, cerebral ischemia, hepatopathy, and the like. Furthermore, there have been reports on many diseases such as diabetes, nervous diseases, renal diseases, hepatic cirrhosis, arthritis, retinopathy of prematurity, ocular uveitis, retinal rust disease, senile cataract, side-effect failures due to radiation therapy, asbestos diseases, bronchial failures due to smoking, anticancer drug side-effect failures, cerebral edema, pulmonary edema, foot edema, cerebral infarction, hemolytic anemia, progeria, spilepsy, Alzheimer disease, Down syndrome, Parkinson disease, Behect's disease, Crohn's disease, Kawasaki disease, Weber-Christian disease, collagen disease, progressive systemic sclerosis, herpetic dermatitis, immune deficiency syndrome, and the like. Although the active oxygen species causing oxidative stress are originally necessary and essential for biological defense, excessive oxidative stress is often present due to reductions of in-vivo antioxidative substances with changes in easting habits or increases in amount of lipids which easily produce release sources of free radicals. According to many researches, it is thought to be nearly certain that the oxidative stress acts as triggers or worsening factors of many diseases. The effectiveness of an antioxidant (radical scavenger) thought to have the ability to eliminate the oxidative stress on the above diseases has studied from old times, and an anti-inflammatory agent exhibiting a radical eliminating ability has been developed. Also, a novel substance (Radicut) exhibiting an antioxidative function has recently been recognized as a medicine. Therefore, it can be said obvious that a substance having an antioxidative function is useful in decreasing oxidative stress. However, such a substance is a medicine, and thus not everybody can use it. Examples of an easily usable substance include antioxidants used for supplements and the like. Namely, the examples include vitamin E, vitamin C, cortisol, β-carotene, vitamin A, BHA (2,6-di-t-butyl-4-methoxyphenol), BHT (2,6-di-t-butyl-4-methylphenol), 7,8-benzoflavone, copper, 3,5-diisopropyl salicylate, and the like. However, the effectiveness of these examples has not yet been known. Conversely, it has been reported that large doses of these antioxidants for increasing the effectiveness produce side effects. For example, with respect to vitamin E, the clinical test results of kidney dialysis patients have recently been reported (Lancet, 356, 1213-1218, 2000). The death rate of kidney dialysis patients by circulatory diseases is 5 to 20 times as high as persons not undergoing kidney dialysis, and oxidative stress is through to be involved as a factor in this result. This report shows the research result that the rate of coincidence of circulatory diseases was decreased by 10% to 20% by administering vitamin E to a patient in a daily dose of 800 IU (80 to 100 times as large as a normal dose). Also, the report shows that a risk of brain hemorrhage as a side effect of vitamin E was increased by 63%, and two patients died from hemorrhagic diseases. In this report, it is described that the rate of coincidence of circulatory diseases due to oxidative stress can be clearly decreased with vitamin E. The report further shows the risk of side effects of vitamin E and discloses that a further research on the use of vitamin E is required from the viewpoint of side effects. With respect to the side effects of large doses of vitamin E, the American Committee of Fetus and Newborn Pediatrics recommended to stop large doses of vitamin E because many newborn babies died in 1985. At the same time, the side effects of oral administration, such as septicemia, necrotizing colitis, and the like, were thought to be due to overdoses of vitamin E. Also, the side effects of intravenous administration were thought to be due to a synergistic action with a surfactant used for suspending vitamin E (Committee of Fetus and Newborn Pediatrics, 76, 315, 1985; D. L. Phelps, Amer. J. Clin. Nutr., 46, 187, 1987). Furthermore, some doubt is cast on the effects of vitamin C and vitamin A in vivo, and the side effects thereof are also suggested (Kunihiko Sato, Jikken Igaku (Experimental Medicine), 4, 1116, 1986; Y. Oyanagi, Biochem. Pharmacol., 25, 1473, 1976). In this way, under the present circumstances, there are many examples in which a substance exhibiting a strong antioxidative activity in vitro does not necessarily exhibit an effective activity in vivo. The great problem of curing with such an antioxidant is that while it can be expected that an antioxidant can decrease oxidative stress to inhibit the occurrence or worsening of a disease, a dose having the probability of showing effectiveness, i.e., a large dose, causes side effects with a high probability. Therefore, an oxidative stress lessening substance (antioxidant) in vivo, which has high safety and can be safely used, has not yet been found.

SUMMARY OF THE INVENTION

[0003] An object of the present invention is to provide an antioxidant with excellent safety, which can lessen oxidative stress in vivo.

[0004] As a result of research for solving the above problem, the inventors found that oxidized coenzyme Q and reduced coenzyme Q with high safety have the antioxidative effect of lessening oxidative stress in vivo.

[0005] Namely, the present invention relates to a composition for lessening oxidative stress, which comprises coenzyme Q as an active ingredient.

DETAILED DISCLOSURE OF THE INVENTION

[0006] A composition for lessening oxidative stress of the present invention comprises, as an active ingredient, coenzyme Q represented by formula (1) and/or formula (2):

[0007] (wherein n represents an integer from 1 to 12);

[0008] (wherein n represents an integer from 1 to 12).

[0009] Coenzyme Q is an essential component which is distributed in a wide variety of living organisms ranging from bacteria to mammals. It is known that coenzyme Q is present as a constituent in the electron transport system of the mitochondria in the organic cells. It is also known that coenzyme Q undergoes oxidation/reduction cycles in the mitochondria to function as a transfer component in the electron transport system, and reduced coenzyme Q exhibits an antioxidative function in vitro. Human coenzyme Q is mainly composed of coenzyme Q₁₀, having 10 repeat structures in its side chain. Although reduced coenzyme Q₁₀ exhibits antioxidative activity in vitro, oxidized coenzyme Q₁₀ exhibits no antioxidative activity. However, oxidized coenzyme Q₁₀ is thought to be converted to a reduced form with a reductase in vivo.

[0010] The high safety of coenzyme Q₁₀ is regarded as its important characteristic. In a chronic toxicity test of rats, it has been reported that no toxic effect was observed in continuous administration in a daily dose of 1200 mg/kg/day for 52 weeks (K. D. Williams, et al. J. Agric. Food Chem., 47, 3756-3763, 1999). The daily dose of 1200 mg/kg/day is converted into a dose of 60 g/day for human beings (body weight 50 kg). Since a usual dose of coenzyme Q₁₀ used as a health food in the United States and Europe is 100 to 300 mg/day, coenzyme Q₁₀ is found to be a supplement material having a high safety rate.

[0011] Coenzyme Q is represented by formula (1) and/or formula (2):

[0012] (wherein n represents an integer from 1 to 12);

[0013] (wherein n represents an integer from 1 to 12) Oxidized coenzyme Q is represented by formula (1) and reduced coenzyme Q is represented by formula (2).

[0014] As shown by the formulae (1) and (2), coenzyme Q having 1 to 12 repeat units (n in each formula) in its side chain can be used in the present invention. Particularly, coenzyme Q having 10 repeat units in its side chain, i.e., coenzyme Q₁₀, can be preferably used.

[0015] The method for producing oxidized coenzyme Q and reduced coenzyme Q is not particularly limited. For example, an adaptable method comprises producing coenzyme Q by a conventional known method such as synthesis, fermentation or extraction from a natural resource, and then concentrating an oxidized coenzyme Q fraction or reduced coenzyme Q fraction of from a chromatography eluate. Oxidized coenzyme Q can be obtained by a known method. On the other hand, when reduced coenzyme Q is desired, a general reducing agent such as sodium borohydride, sodium dithionite (sodium hydrosulfite), or the like may be added to the coenzyme Q according to demand to reduce the coenzyme Q to reduced coenzyme Q by a conventional method, and then the reduced coenzyme Q may be concentrated by chromatography. Also, reduced coenzyme Q can be obtained by a method in which existing high-purity coenzyme Q is reacted with a reducing agent.

[0016] In the present invention, a reduced form and an oxidized form may be singly used as coenzyme Q, or a mixture of the oxidized form and reduced form may be used. In the use of coenzyme Q₁₀, a mixture of the reduced form and oxidized form has higher oral absorptivity than that of the use of only the oxidized form (Publication WO98/07417), and thus coenzyme Q₁₀ containing the reduced form is preferably used as an oral agent. In this case, from the viewpoint of oral absorptivity the ratio of reduced coenzyme Q₁₀ in the total of coenzyme Q₁₀ is preferably 20% by weight or more, more preferably 40% by weight or more, and most preferably 60% by weigh or more. Although the upper limit of the ratio is not limited, the upper limit is generally 99% by weight or less.

[0017] Although the total content of coenzyme Q in the composition of the present invention is not particularly limited, the content is preferably from 0.001% by weight to 99% by weight, and more preferably from 0.01% by weight to 20% by weight, from the viewpoint of effectiveness.

[0018] Besides the coenzyme Q, the composition of the present invention may contain various additives allowable in the medical field or Food Sanitation Law. When the composition is used as a countermeasure against one of various diseases, the composition can be used in a combination with a medicine for a disease. Furthermore, another antioxidant, a health food material, a supplement material and vitamin may be added. Since reduced coenzyme Q is known to have the ability to reproduce vitamin E, a combination of reduced coenzyme Q and vitamin E is expected to show a synergistic function.

[0019] The administration form of the composition of the present invention may be a liquid or a solid, and the administration method may be any one of various methods such as oral administration, administration of an injection, nasal drops, eye drops, or a suppository, eating of coenzyme Q-containing food, and the like. Generally, oral administration is though to be effective from the viewpoint of dosage. However, when the composition is used for a local disease or when oral administration is difficult, there is no problem in any administration method of the composition of the present invention, other than oral administration. Conceivable examples of such an administration method include administration of eye drops for preventing diabetic retinopathy, administration of a suppository to a patient, an elderly person or an infant who has a difficulty in oral administration of nutrients, administration of an external preparation for skin diseases, and the like. However, the administration method is not limited to these examples.

[0020] The composition of the present invention can be produced by adding coenzyme Q as the active ingredient, and adding various additives according to demand.

[0021] In the oral administration of the composition of the present invention, the dose of coenzyme Q per kg of weight of a human body is preferably from 0.1 mg/day to 500 mg/day, more preferably from 1 mg/day to 100 mg/day, and most preferably from 2 mg/day to 75 mg/day.

[0022] The composition of the present invention has the antioxidative function to lessen oxidative stress in vivo. The in-vivo antioxidative function of coenzyme Q was discovered in the present invention for the first time. Therefore, the composition of the present invention is effective in curing and/or preventing diseases due to oxidative stress.

[0023] As described above, coenzyme Q₁₀, which is a type of enzyme Q, has high safety, and thus the in-vivo use of the composition containing coenzyme Q₁₀ has no danger of side effects. This property is not exhibited by vitamin E known to have the same antioxidative function, and thus the property can be regarded as an excellent characteristic of the composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a bar graph showing the amounts of urinary 8OH-dG of SD rats administered with reduced or oxidized coenzyme Q₁₀, in which the amount of urinary 8OH-dG is shown as ordinate, data of average ±SD with n=3 is shown, and * shows significance against a control group with a risk rate of 5% in a Student t-test carried out as a significant difference test.

[0025]FIG. 2 is a bar graph showing the amounts of urinary 8OH-dG of GK rats administered with reduced or oxidized coenzyme Q₁₀, in which the amount of urinary 8OH-dG is shown as ordinate, data of average ±SD with n=4 is shown. In the figure, Cont. represents a control group, Red. represents a reduced coenzyme Q₁₀ administration group, Ox represents an oxidized coenzyme Q₁₀ administration group, and VE represents a vitamin E administration group. In the graph, values measured 2 weeks after the administration are shown in the left half, and values measured 4 weeks after the administration are shown in the right half. In the graph, ** and * show significance against a control group with risk rates of 1% and 5%, respectively, in a Student t-test carried out as a significant difference test.

[0026]FIG. 3 is a bar graph showing the amounts of urinary 8OH-dG of KK-Ay mice administered with reduced or oxidized coenzyme Q₁₀ in which the amount of urinary 8OH-dG is shown as ordinate, data of average ±SD with n=11 is shown, and * shows significance against a control group with a risk rate of 5% in a Student t-test carried out as a significant difference test.

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] Although the present invention will be described in further detail below with reference to examples, the present invention is not limited to these examples.

[0028] The effect of lessening oxidative stress in vivo was evaluated with urinary 8-hydroxydeoxyguanosine (8OH-dG) having high effectiveness as an oxidative stress marker. This material results from nucleic acid damage due to oxidative stress, and is discharged into urine without undergoing further metabolism. It is thus thought that the amount of oxidative stress in vivo and the amount of 8OH-dG discharged have a close relationship therebetween. A decrease in amount of the material in urine means a reduction in oxidative stress in vivo and the prevention of damage to nucleic acid.

EXAMPLE 1 Oxidative Stress Lessening Effect on Normal Rat

[0029] A soybean oil solution of oxidized coenzyme Q₁₀ or reduced coenzyme Q₁₀ (containing about 2% of oxidized coenzyme Q₁₀) was orally administered to SD rats (6-week old, male) in a dose of 100 mg/kg/day for 4 weeks. 4 weeks after, urine was collected, and the amount of urinary 8OH-dG was determined by an ELISA kit (Japan Institute for the Control of Aging). The results are shown in FIG. 1. In the oxidized coenzyme Q₁₀ administration group, the amount of urinary 8OH-dG was significantly decreased to about 60% of that of the solvent control group (urinary 8OH-dG was assumed as 100%). In the reduced coenzyme Q₁₀ administration group, the amount of urinary 8OH-dG was significantly decreased to about 40% of that of the solvent control group.

EXAMPLE 2 Oxidative Stress Lessening Effect on Spontaneously Diabetic Rat

[0030] The oxidative stress lessening effect of each of reduced and oxidized coenzyme Q₁₀ in vivo was evaluated by using GK rats with spontaneous diabetes which induced spleen tissue denaturation due to oxidative stress in vivo. A feed (CE-2, produced by CLEA JAPAN, INC.) containing 0.1% by weight of reduced coenzyme Q₁₀ (containing about 2% by weight of oxidized coenzyme Q₁₀) or oxidized coenzyme Q₁₀ was freely given to GK rats (5-week old, male, n=4 in each group). In a control group, the feeds not containing coenzyme Q₁₀ was freely given to the rats. 2 weeks and 4 weeks after, urine was collected, and the amount of urinary 8OH-dG was determined by the same method as in Example 1. The results are shown in FIG. 2. In the reduced coenzyme Q₁₀ administration group, the amount of urinary 8OH-dG measured 2 weeks after the administration was significantly decreased to about 40% of that of the control group (urinary 8OH-dG was assumed as 100%), and the amount of urinary 8OH-dG measured 4 weeks after the administration was kept at the same level. In the oxidized coenzyme Q₁₀ administration group, the amount of urinary 8OH-dG measured 2 weeks after the administration was decreased to 58% of the control group, and the amount of urinary 8OH-dG measured 4 weeks after the administration was deceased to 52% of the control group. Although the amounts of urinary 8OH-dG in the oxidized coenzyme Q₁₀ administration group were larger than those in the reduced coenzyme Q₁₀ administration group, the amounts of urinary 8OH-dG were significantly decreased in comparison to the control group.

REFERENCE EXAMPLE 1 Oxidative Stress Lessening Effect of Vitamin E on Spontaneously Diabetic Rat

[0031] The oxidative stress lessening effect of a typical antioxidant, vitamin E, was evaluated by using GK rats in the same test system as in Example 2. Like coenzyme Q₁₀, vitamin E was mixed with a feed at a ratio of 0.1% by weight, and the resulting foodstuff was freely given to the GK rats. 2 weeks and 4 weeks after the feeding, the amount of urinary 8OH-dG was determined. The results are shown in FIG. 2. In the vitamin administration group, the amount of urinary 8OH-dG measured 2 weeks after the administration was decreased to about 80% of that of the control group, and only a downward tendency was exhibited. The amount of urinary 8OH-dG measured 4 weeks after the administration was significantly decreased to 51%. In comparison to the coenzyme Q₁₀ administration group of Example 2, 2 weeks after, the decreasing effect in Reference Example 1 was lower the effects of both the reduced form and the oxidized form in Example 2, and 4 weeks after, the effect was at the same level as that of the oxidized coenzyme Q₁₀ and lower than that of the reduced coenzyme Q₁₀ in Example 2. These results indicate that vitamin E requires much time for expressing antioxidative activity, as compared with coenzyme Q₁₀.

EXAMPLE 3 Oxidative Stress Lessening Effect on Spontaneously Diabetic Mouse

[0032] The dose dependency of the oxidative stress lessening effect of each of reduced and oxidized coenzyme Q₁₀ was evaluated by the same method as that used for GK rats except that KK-Ay mice with spontaneous diabetes and having a higher blood sugar level than that of GK rats were used. KK-Ay 5-week old male mice were used, and evaluated in groups in each of which n=11. Oxidized coenzyme Q₁₀ or reduced coenzyme Q₁₀ (containing about 2% by weight of oxidized coenzyme Q₁₀) was mixed with a purified feed (produced by CLEA JAPAN, INC.) at each of ratios of 0.02% by weight, 0.1% by weight, and 0.5% by weight, and each mixture was freely given to the KK-Ay mice. In a control group, the purified feed not containing coenzyme Q₁₀ was freely given to rats. 4 weeks after the supply, urine was collected, and the amount of urinary 8OH-dG was determined by the same method as in Example 1. The results are shown in FIG. 3. Both the reduced coenzyme Q₁₀ and the oxidized coenzyme Q₁₀ exhibited the dose-dependent oxidative stress lessening effect, as compared with the control group. In the reduced coenzyme Q₁₀ administration group, with a dose of 0.02% by weight, the lessening effect was low (82% of the control group), while with a dose of 0.1% by weight or more, the lessening effect was significant (dose of 0.1% by weight: 73% of the control group, dose of 0.5% by weight: 56% of the control group). In the oxidized coenzyme Q₁₀ administration group, the dose-dependent lessening effect was observed without showing a significant difference, and with a dose of 0.5% by weight, the amount of urinary 8OH-dG was 78% of that of the control group.

[0033] As described above, the oxidative stress lessening effect of coenzyme Q₁₀ in vivo was confirmed with normal rats, spontaneously diabetic rats and spontaneously diabetic mice. Also, in any one of the tests, the effect of the reduced form was higher than that of the oxidized form, and the effectiveness of reduced coenzyme Q₁₀ is expected.

[0034] Also, the GK rats used in Example 2 are known to have the spleen tissue damaged by oxidative stress, and thus evaluation was made for the protective effect of decreasing oxidative stress by coenzyme Q₁₀ to protect GK rats from denaturation of the spleen tissues.

EXAMPLE 4 Tissue Protecting Effect on Spontaneously Diabetic Rat

[0035] The protective effect of each of oxidized coenzyme Q₁₀ and reduced coenzyme Q₁₀ (containing about 2% by weight of oxidized coenzyme Q₁₀) on denaturation of the spleen tissues of GK rats by using the same test system as in Example 1. As a control, a soybean oil solution not containing coenzyme Q₁₀ was orally administered. After the coenzyme Q₁₀ was administered for 7 weeks, the spleen was extracted from each rat, and the degree of tissue denaturation was histopathologically evaluated. The results are shown in Table 1. TABLE 1 Number of examples Reduced Oxidized coenzyme coenzyme Findings Control Q₁₀ Q₁₀ VE Normal 0 0 0 0 Islet fibrillation 1+ 0 3 2 2 Islet fibrillation 2+ 4 1 2 2

[0036] The fibrillation of the Langerhans' islet tissue known to secrete insulin was observed for the spleens. In the control group, fibrillation at a degree of +2 (higher than a degree of +1) was observed in all of the four examples. In the reduced coenzyme Q₁₀ administration group, fibrillation at a degree of +1 was observed in tree examples, and fibrillation at a degree of +2 was observed in one example. Namely, the tendency to decrease fibrillation was observed. In the oxidized coenzyme Q₁₀ administration group, fibrillation at a degree of +1 was observed in two examples, and fibrillation at a degree of +2 was observed in two examples. Namely, the tissue denaturation due to oxidative stress was decreased, as compared with the control group. However, the protective effect of the oxidized coenzyme Q₁₀ administration group was slightly lower than that of the reduced coenzyme Q₁₀ administration group.

REFERENCE EXAMPLE 2 Tissue Protecting Effect of Vitamin E on Spontaneously Diabetic Rat

[0037] The tissue protecting effect of vitamin E was evaluated by the same method as in Example 4. As a result, fibrillation at a degree of +1 was observed in two examples, and fibrillation at a degree of +2 was observed in two examples, as shown in Table 1. Therefore, the results were the same as oxidized coenzyme Q₁₀, and tissue denaturation was decreased in comparison to the control group. However, the tissue protecting effect of vitamin E was slightly lower than that of the reduced coenzyme Q₁₀ administration group.

[0038] As a result of the evaluation of the protective effect of each of oxidized coenzyme Q₁₀ and reduced coenzyme Q₁₀ on denaturation of the spleen tissues of the GK rats, it was found that tissue denaturation is decreased by administering coenzyme Q₁₀, and the protective function of reduced coenzyme Q₁₀ is higher than that of oxidized coenzyme Q₁₀. The protective effect on fibrillation of the spleen Langerhans' islet tissue of a GK rat is caused by a decrease in oxidative stress due to oxidized coenzyme Q₁₀ and reduced coenzyme Q₁₀ in vivo. The result indicates that coenzyme Q₁₀ is useful as a substance (antioxidant) for lessening oxidative stress in vivo. As a result of the evaluation of vitamin E which is a typical existing antioxidant, and comparison to coenzyme Q₁₀, the activity to decrease the amount of 8OH-dG and the activity to protect the spleen tissue from fibrillation were observed. However, both activities of vitamin E were at the same level as or lower than those of oxidized coenzyme Q₁₀, and were clearly lower than those of reduced coenzyme Q₁₀.

[0039] As described above, vitamin E uncertainly exhibits effectiveness for human beings. However, the possibility of the side effects increases as the dose increases, thereby causing difficulty in achieving a secured effect. On the other hand, the high safety of coenzyme Q₁₀ was proved, and coenzyme Q₁₀ exhibits an in-vivo antioxidative activity equal to or higher than that of vitamin E and significantly decreases oxidative stress in vivo. It is thus decided that coenzyme Q₁₀ is an antioxidant useful for various diseases caused by oxidative stress or worsened by oxidative stress.

[0040] Industrial Applicability

[0041] The composition of the present invention comprising coenzyme Q as an active ingredient decreases oxidative stress in vivo, and exhibits an excellent effect on many diseases and health maintenance. 

1. A composition for lessening oxidative stress, comprising, as an active ingredient, coenzyme Q represented by formula (1) and/or formula (2):

(wherein n represents an integer from 1 to 12);

(wherein n represents an integer from 1 to 12).
 2. The composition according to claim 1, wherein the coenzyme Q is coenzyme Q₁₀.
 3. The composition according to claim 1, wherein the coenzyme Q content is from 0.001% by weight to 99% by weight. 